Despite well established roles of ROS/RNS in alcohol-induced cell injury, the proteins that are selectively oxidized by ROS/RNS are poorly characterized. We hypothesized that certain cysteine residues of the target proteins are oxidized by ROS upon alcohol exposure, and these modified proteins may play roles in alcohol-mediated mitochondrial dysfunction and cell damage. A targeted proteomics approach using biotin-N-maleimide (biotin-NM) as a specific probe to label oxidized proteins has been developed at our laboratory and used to identify oxidized mitochondrial proteins during and after alcohol exposure. Human hepatoma HepG2 cells with transduced CYP2E1 (E47 cells) were used as a model to generate ROS through CYP2E1-mediated ethanol metabolism. Following exposure to 50 and 100 mM ethanol for 4 and 8 hours, increased levels of biotin-NM-labeled proteins were detected and oxidized proteins subsequently purified with agarose-streptavidin or agarose-monoclonal antibody against biotin. The purified oxidized proteins were resolved by 2-D gel electrophoresis and protein spots that displayed differential abundances were excised from the gel, in-gel digested with trypsin and subjected to mass spectrometry. The mass spectrometric analysis revealed that many mitochondrial proteins including mitochondrial aldehyde dehydrogenases were oxidized after alcohol exposure. Because of the time differences in protein modification (observed at early times) and apoptosis (at later times) after alcohol exposure, we believe that inactivation or functional loss of some of these oxidized proteins may contribute toward ethanol-mediated oxidative injury. This method was also used to identify oxidized mitochondrial and cytosolic proteins isolated from alcohol-fed mouse liver to demonstrate the utility of our detection method for in vivo tissue samples. Our unpublished results of mass spectrometric analysis show that many cytosolic proteins, involved in the efficient removal of peroxides (e.g. peroxiredoxin isozymes), glutathione biosynthesis and utilization, and cellular protective enzymes (heat shock proteins, aldehyde dehydrogenase, isocitrate dehydrogenase, etc), were oxidized after chronic alcohol exposure. The mechanism of inactivation of peroxiredoxin isozymes and its reactivation by sulfiredoxin or sestrins is being investigated in E47 cells after acute ethanol exposure. We plan to measure the activities of individual enzymes, since potential inactivation of these enzymes through oxidation of certain critical cysteine residues may provide the molecular basis for the well-established results of reduced glutathione levels and increased peroxide levels after alcohol exposure. Our current detection method has clear advantages over other existing methods for detecting oxidized proteins and can also be applied to identification of oxidized proteins in another type of cultured cells, subcellular fractions, or animal tissues exposed to various chemicals known to produce ROS/RNS or under pathophysiological conditions. We have recently reported persistent activation of c-Jun N-terminal protein kinase (JNK) and p38 protein kinase (p38 kinase) by many substrates of CYP2E1 such as ethanol, acetaminophen (APAP), 4-hydroxynonenal, carbon tetrachloride, and long chain fatty acids as well as a non-CYP2E1 substrate such as troglitazone, which causes liver damage. Our unpublished results also showed that ethanol caused time- and dose-dependent cell death in E47 HepG2 cells and two other types of cells in culture. Ethanol increased the activities of JNK and p38 kinase and caused translocation of proapoptotic Bax to mitochondria in a time-dependent manner. Activation of both JNK and p38 kinase seemed important in ethanol-induced cell death, because pretreatment with a respective inhibitor of JNK or p38 kinase significantly reduced the activity of each kinase and the rate of ethanol-induced apoptosis.. Based on these results, we hypothesized that proapoptotic Bax protein is retained by its anchoring proteins in the cytosol under physiological resting states but Bax may be unleashed from its anchoring proteins and translocate to mitochondria after exposure to cell death stimuli, most of which activate JNK and p38 kinase. We have so far identified several cytosolic proteins that could bind Bax and thus potentially prevent Bax translocation to mitochondria prior to cytochrome c release, caspase activation, and apoptosis. We are currently determining the binding and dissociation kinetics between Bax and its anchoring proteins. We have started investigating the direct relationship between activated JNK or p38 kinase and Bax or its anchoring proteins in alcohol-treated E47 HepG2 cells. Our results so far seem to represent new data for better understanding about the role of Bax anchoring proteins in Bax-mediated apoptosis. Our results also reflect the true Bax anchoring proteins present under normal physiological states. These proteins are different from those recently identified under non-physiological systems with artificially transfected DNA coding for a specific protein of interest.